PRODUCTION OF SOMATOMEDIN ACTIVITY IN VITRO IN THE PRESENCE OF GROWTH HORMONE AND CYCLOHEXIMIDE

J. P. LIBERTI Medical Department of Biochemistry, College of Virginia, Virginia Commonwealth Richmond, Virginia 23298, U.S.A. University,

(Received 13 February 1978) McConaghey & Sledge (1970) first demonstrated the production of somatomedin after perfusion of livers from normal rats with growth hormone (GH). Subsequent work has firmly established that the liver is an important site of somatomedin production and that GH plays an active role in this process (McConaghey, 1972; Uthne & Uthne, 1972; Phillips, Herrington, Karl & Daughaday, 1976), although little is known concerning its mechanism of action. Initially, several mechanisms, which can be discriminated in part by the requirement for active protein synthesis, appear possible. One of these states that GH, or perhaps a catabolite of it, stimulates the liver to synthesize somatomedin de novo and hence agents which inhibit protein synthesis could abolish the effects of GH on somatomedin production. The experiments reported here represent our initial attempts to test this hypothesis. Tissues were obtained from hypophysectomized rats weighing 100-120 g (Zivic-Miller Labs, Allison Park, U.S.A.). Hepatocytes, prepared as described by Howard & Pesch (1968), were dispersed in Eagle's medium containing Earle's salts, 500 u. penicillin and 500 µg streptomycin (Flow Laboratories). Bovine GH (NIH-GH-B-16) was dissolved in 0-1 M-NaOH and diluted with 0-9% saline ; solutions of cycloheximide in saline containing 2% ethanol were prepared freshly for each experiment. Carrier-free Na235S04 and [3H]leucine were purchased from New England Nuclear (Boston, Massachusetts, U.S.A.). Portions (2 ml) of the hepatocyte suspension were incubated with GH alone or in the presence of cycloheximide at 22 °C (cells appeared to be more responsive to GH at 22 than at 37 °C); control flasks contained an equal volume of the diluent used to prepare the GH and cycloheximide solutions. After incubation for 2 h, the cells were pelleted by centrifugation and 1 ml of each supernatant fraction was passed through a column of Sephadex G-15 (1-5 cm 8 cm) at 22 °C, with 0-5 M-KC1 as eluant, at a flow rate of 30 ml/h. Portions of the material eluting in the void volume were assayed for sulphation activity. Sulphation, i.e. somatomedin-like activity, was determined by measuring the uptake of 35S042- by costal cartilage (Salmon & Daughaday, 1957). Each flask contained six cartilage segments, 1-25 µ Na235S04, 2 ml Eagle's medium (as above), 0-34 mM-serine and a control sample or portion of test solution. After incubation for 16 h at 37 °C, the reaction was stopped by boiling and the segments were washed, dried, weighed and digested in 88% formic acid. The radioactivity was determined by liquid scintillation counting (Bray, 1960); significances were determined by Student's /-test. The validity of the assay was assessed serum samples from normal and hypopituitary subjects ; a linear response relating the uptake of 35S042- and the log of the dose was found with both sera. The potency of hypo¬ pituitary dwarf serum was 60% of the potency of normal serum, which agrees well with results reported by others (Hall, 1971). The results in Table 1 show that incubation of GH in medium containing no hepatocytes had no stimulatory effect on sulphation activity. The uptake of ^SO^- was stimulated by with

Table 1. Somatomedin activity of rat hepatocytes after incubation with GH(2·4 10~ alone or in the presence of cycloheximide (0-5 mmol/l) Additions to incubation

cycloheximide diluent, hepatocytes GH, hepatocytes GH, cycloheximide, hepatocytes GH, no hepatocytes

GH

Uptake of Na235S04 (counts min-2 mg cartilage-1)*

% of control

7315 ±556 9050 ±434

100 132

11660±1283 7632 ±611

159 104

value

mol/ï)

Pf

or

Production of somatomedin activity in vitro in the presence of growth hormone and cycloheximide.

PRODUCTION OF SOMATOMEDIN ACTIVITY IN VITRO IN THE PRESENCE OF GROWTH HORMONE AND CYCLOHEXIMIDE J. P. LIBERTI Medical Department of Biochemistry, Col...
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